Cutaneous Biology of KIT Ligand

KIT 配体的皮肤生物学

• 批准号: 6755162
• 负责人: B Jack Longley
• 金额: $ 31.1万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-01-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6755162
• 关键词: biopsy; cell proliferation; contact dermatitis; family genetics; gene mutation; genetic mapping; genetic promoter element; genetically modified animals; human subject; inflammation; laboratory mouse; loss of heterozygosity; mastocytosis; neoplastic transformation; nucleic acid sequence; pathologic process; patient oriented research; phosphatidylinositol 3 kinase; protein tyrosine kinase; skin disorder; stem cell factor; tissue /cell culture

DESCRIPTION (provided by applicant): Stem Cell Factor (SCF, also known as mast cell growth factor), is the ligand for KIT, a receptor tyrosine kinase. The goal of this proposal is to determine the role of the SCF-KIT signaling pathway in mastocytosis and cutaneous inflammation. Mastocytosis occurring sporadically in adults is caused by somatic mutations affecting the primary sequence of KIT and causing constitutive activation of KIT and its downstream transducing molecule PI3-K, which causes phosphorylation of AKT. Familial and most pediatric cases of mastocytosis cases show normal KIT protein coding sequence but have phosphorylated AKT in lesional mast cells. Our first hypothesis is that familial and sporadic pediatric mastocytosis are caused by mutations affecting the SCF-KIT signaling pathway, or pathways convergent with it at or above AKT. Human epidermal keratincytes produce SCF, and dermal injection of SCF causes inflammation. Trangenic mice which express epidermal SCF, like humans, show an exaggerated ear swelling response to allergic and irritant contactants. Our second hypothesis is that SCF-KIT signaling plays an active role in the cutaneous inflammatory response. Our specific aims are: 1. To determine the mechanism(s) of oncogenesis in c-KIT mutation negative pediatric mastocytosis, mRNA from lesional mast cells will be RT-PCR amplified and sequenced to detect mutations in genes encoding molecules which may affect AKT phosphorylation including AKT, PTEN, Lyn and PI3-K. Since loss of PTEN could result in increased PI3-K signaling, lesional mast cell DNAk will also be tested for loss of heterozygosity in region 10q23 by microsatellite analysis. The functional effects of mutations or gene loss will be determined in cultured bone marrow derived mast cells an mast cell lines by retroviral expression of mutant activating or dominant negative proteins, or by anti-sense suppression. 2. To determine the genetic basis of familial mastocytosis, two separate kindreds with dominantly inherited mastocytosis will be tested for linkage to genes known to affect the KIT-P13-K signaling pathway using microsatellite analysis. If necessary, a genome-wide screen of affected and genetically relevant unaffected individuals will be performed using loci at 10 cM intervals followed by positional cloning and gene identification. 3. To test the hypothesis that SCF-KIT signaling is actively involved in the afferent, efferent, or both arms of the cutaneous immune response, we will use adoptive transfer of immune lymphocytes, KIT blocking antibodies, and small molecule inhibitors of KIT in a series of DNFGB sensitivity studies in normal mice, and in a proven transgenic model of SCF-KIT mediated cutaneous inflammation. These studies will determine specific contributions of SCF-KIT signaling to contact dermatitis, an provide support for the hypothesis that inhibitors of KIT may be novel therapeutic agents for human cutaneous inflammation.

描述（由申请人提供）：干细胞因子（SCF，也称为桅杆 细胞生长因子），是一种受体酪氨酸激酶试剂盒的配体。这 该建议的目标是确定SCF-KIT信号通路的作用 在肥大和皮肤炎症中。 成人偶尔出现的肥大性发生是由体细胞突变引起的 影响试剂盒的主要序列，并引起构成激活 套件及其下游转导的分子PI3-K，它导致磷酸化 Akt。家族性和大多数小儿肥大病例病例表明套件正常 蛋白质编码序列，但在病变肥大细胞中磷酸化AKT。我们的 第一个假设是家族性和零星的小儿肥大性是 由影响SCF-KIT信号通路或途径的突变引起 在Akt或之上收敛。 人表皮角膜细胞产生SCF，并注入SCF原因 炎。像人类一样表达表皮SCF的眨眼小鼠 对过敏和刺激性接触剂的夸大耳朵肿胀反应。我们的 第二个假设是SCF-KIT信号在 皮肤炎症反应。我们的具体目的是：1。确定 C-KIT突变阴性小儿肥大症中肿瘤发生的机制， 病变肥大细胞中的mRNA将得到RT-PCR的扩增并测序以检测 编码可能影响Akt磷酸化的分子的基因突变 包括AKT，PTEN，LYN和PI3-K。由于失去PTEN可能会导致 PI3-K信号传导增加，病变肥大细胞DNAK也将被测试是否损失 通过微卫星分析，区域10q23中的杂合性。功能 突变或基因丧失的影响将在培养的骨髓中确定 通过突变体逆转录病毒表达衍生的肥大细胞肥大细胞系 激活或显性的负蛋白，或通过抗沉抑抑制。 2 确定家族性肥大症的遗传基础，两种单独的亲属 将对遗传性肥大遗传学作用进行测试，以与基因联系 已知会使用微卫星分析影响Kit-P13-K信号通路。 如有必要，全基因组屏幕受影响和遗传相关的 未受影响的个体将在10 cm的间隔内使用基因座进行 通过位置克隆和基因鉴定。 3。检验以下假设 SCF-KIT信号积极参与传入，屈从或两个臂 在皮肤免疫反应中，我们将使用免疫的收养转移 淋巴细胞，试剂盒阻断抗体和小分子抑制剂在A中 在正常小鼠中的一系列DNFGB敏感性研究，并在经过验证的转基因中 SCF-KIT介导的皮肤炎症的模型。这些研究将确定 SCF-KIT信号对接触皮炎的特定贡献，提供 支持试剂盒抑制剂可能是新型治疗的假设 人皮肤炎症的药物。